Autotransporter (AT) proteins represent the largest family of virulence proteins secreted from Gram-negative bacterial pathogens. Each monomeric AT has the same core components for translocation and virulence, including a β-helical passenger domain that comprises the mature, functional protein. Proteins with β-helix structure have neither been found in mammalian proteomes nor in commensal microbes and thus represent an attractive target for the development of novel antimicrobial agents. Despite having the same secretion mechanism and basic structure, ATs have diverse functions including cell adhesion and invasion of host cells. Our lab uses pertactin from Bordetella pertussis as a model to study AT secretion and folding. We previously showed that the pertactin passenger folds as it is translocated across the outer membrane (OM) and that a pertactin mutant that cannot fold leads to a ∼10-fold reduction in secretion efficiency. Furthermore, inducing AT passengers to adopt stable structure in the bacterial periplasm can block their translocation across the OM. The precise mechanism by which pertactin and other ATs remain in a high energy, non-native state in the periplasm and yet efficiently fold to the native structure at the cell surface remains unknown. We have converted a low-throughput, western blot-based assay for quantifying AT OM translocation efficiency into two high-throughput fluorescence assays. The first is a flow cytometry assay for testing the impact of a library of pertactin mutants on OM translocation efficiency, to determine which residues are most important. The second is a 96-well plate-based assay for identifying small molecules that disrupt AT OM translocation. Because the β-helix is a distinct—and ubiquitous—feature of Gram-negative bacterial pathogenesis, we anticipate that these assays will enable us to identify key vulnerabilities for pathogenesis across a wide range of Gram-negative infectious agents, and how they might be exploited.
Patel et al. (Sun,) studied this question.